Tracer kinetic model of blood-brain barrier transport of plasma protein-bound ligands. Empiric testing of the free hormone hypothesis.

Previous studies have shown that the fraction of hormone or drug that is plasma protein bound is readily available for transport through the brain endothelial wall, i.e., the blood-brain barrier (BBB). To test whether these observations are reconcilable with the free-hormone hypothesis, a tracer-kinetic model is used in the present investigations to analyze in vivo initial extraction data on BBB transport of protein-bound steroid hormones (dihydrotestosterone, testosterone, estradiol, and corticosterone), thyroid hormones (triiodothyronine), and lipophilic amine drugs (propranolol). The plasma proteins used are bovine albumin and human orosomucoid. Transport data was fit to a modification of the Kety-Renkin-Crone equation of capillary physiology; the modified equation incorporates the principles of both capillary physiology and plasma protein-ligand mass action binding relationships. In most cases, the experimental data is best fit to the model equation when the apparent in vivo dissociation constant, KDa, of the ligand protein binding reaction increases to values that are 5- to 50-fold greater than the in vitro dissociation constant, KD. This result indicates that the rate of ligand dissociation from the plasma protein is accelerated in the capillary bed relative to the in vitro situation. It is hypothesized that the major factor leading to the rapid transport in vivo of protein-bound ligands into tissues such as brain is an endothelial-induced decrease in the affinity of the plasma protein for the ligand. Under these conditions, the amount of plasma ligand available for tissue clearance in vivo parallels the protein-bound fraction, not the free hormone.

Small molecule inhibition of cAMP response element binding protein in human acute myeloid leukemia cells.

The transcription factor CREB (cAMP Response-Element Binding Protein) is overexpressed in the majority of acute myeloid leukemia (AML) patients, and this is associated with a worse prognosis. Previous work revealed that CREB overexpression augmented AML cell growth, while CREB knockdown disrupted key AML cell functions in vitro. In contrast, CREB knockdown had no effect on long-term hematopoietic stem cell activity in mouse transduction/transplantation assays. Together, these studies position CREB as a promising drug target for AML. To test this concept, a small molecule inhibitor of CREB, XX-650-23, was developed. This molecule blocks a critical interaction between CREB and its required co-activator CBP (CREB Binding Protein), leading to disruption of CREB-driven gene expression. Inhibition of CBP-CREB interaction induced apoptosis and cell-cycle arrest in AML cells, and prolonged survival in vivo in mice injected with human AML cells. XX-650-23 had little toxicity on normal human hematopoietic cells and tissues in mice. To understand the mechanism of XX-650-23, we performed RNA-seq, ChIP-seq and Cytometry Time of Flight with human AML cells. Our results demonstrate that small molecule inhibition of CBP-CREB interaction mostly affects apoptotic, cell-cycle and survival pathways, which may represent a novel approach for AML therapy.

Postremission chemotherapy for adults with acute myelogenous leukemia: improved survival with high-dose cytarabine and daunorubicin consolidation treatment.

Results of postremission chemotherapy for adults with acute myelogenous leukemia (AML) were assessed in two sequential prospective studies involving similar induction therapy and two courses of intensive consolidation treatment. Fifty-six patients achieving remission on the acute leukemia protocol (ALP3) study received high-dose cytarabine and daunorubicin as course one and standard-dose cytarabine and daunorubicin as course two. Results are compared with forty-six patients achieving remission on the ALP2 study who received azacitidine and doxorubicin as consolidation course one and standard-dose cytarabine, daunorubicin, and thioguanine as course two. The ALP3 regimen resulted in a significantly improved 5-year disease-free survival of 32% +/- 19% versus 20% +/- 11% for the ALP2 study (P = .03). Survival from remission was also improved, 40% +/- 14% versus 24% +/- 12% (P less than .01). Favorable prognostic factors for disease-free survival included receiving the ALP3 treatment regimen, absence of a prior preleukemic syndrome, and female sex. These factors and younger patient age were significant for survival following first chemotherapy and survival after achieving remission. Six of 34 patients who relapsed after receiving the ALP3 regimen successfully achieved prolonged second remissions with high-dose cytarabine-based chemotherapy and/or allogeneic bone marrow transplantation (BMT). Overall survival for adults less than or equal to 45 years of age was 58% +/- 19% with the ALP3 postremission chemotherapy regimen, comparable to most studies of BMT for AML in first remission. Actuarial 5-year survival for ALP3 patients greater than 60 years of age was 18% +/- 20% with no improvement compared with ALP2.

Suramin in hormone-refractory metastatic prostate cancer: a drug with limited efficacy.

PURPOSE: To confirm the previously reported high response rates and prolonged survival in hormone-refractory prostate cancer treated with suramin. PATIENTS AND METHODS: Thirty-six eligible patients with hormone-refractory prostate cancer with either measurable disease or bone disease only and a prostate-specific antigen (PSA) level greater than 50 ng/mL were enrolled. Treatment consisted of two 8-week courses of outpatient-based therapy with an interposed rest period. A bayesian adaptive control strategy and a three-compartment pharmacokinetic model that accommodates clearance changes was used to guide individual dosing. A rapid infusion of 1,000 mg/m2 suramin was followed by five daily infusions that targeted 285 micrograms/mL peak plasma levels during the first week. All patients received concomitant hydrocortisone. For the next 7 weeks, patients received one to two doses per week that targeted levels in the 150 to 285 micrograms/mL range and integrated weekly averages of 200 ug/mL. RESULTS: Nine patients (28%) had a partial response to suramin based on a > or = 50% decrease in PSA levels coupled with either relief of bone pain or by a 50% decrease in measurable disease. The median overall survival time for all patients is 31 weeks (95% confidence interval [CI], 23 to 51). Treatment was generally well tolerated, with fatigue being the most common significant toxicity, but fatal idiosyncratic myelosuppression (grade V) was observed in one patient. CONCLUSION: Using this dosing schedule, suramin has limited activity against hormone-refractory metastatic prostate cancer. Recent data suggest that hydrocortisone administered with suramin may be partly responsible for the benefit attributed to the drug. Although a small cohort of patients appeared to benefit, we were unable to confirm the previously reported high rate of activity and durability of remission using this agent.

Renal clear-cell carcinoma: an ultrastructural study on the junctional complexes.

Junctional complexes such as tight junctions, adherens junctions, and desmosomes play crucial roles in the structure and function of epithelial cells. These junctions are involved in increasing cell-cell contact and as well serve as signaling centers regulating multiple functions in epithelial cells. Carcinoma cell lines cultured in the laboratory generally lack junctional complexes. However, studies directed towards understanding the distribution of junctional complexes in human cancer tissues are lacking. In this study, we analyzed by electron microscopy the distribution of junctional complexes in patients diagnosed with renal clear-cell carcinoma. We found that both tight junctions and adherens junctions were drastically reduced in patients with cancer compared to normal tissues. Desmosomes were not detected in normal proximal tubules while distinctly present in cancer tissues. These results suggest that analysis of junctional complexes in human tumors should provide valuable information that might have prognostic and diagnostic value.

Steady state model of 3,5,3'-triiodothyronine transport in liver predicts high cellular exchangeable hormone concentration relative to in vitro free hormone concentration.

The hepatic nuclear receptor for T3 is known to be approximately 50% saturated in vivo. Since the receptor dissociation constant (Kd) is 1 nM, and since 50% receptor occupancy occurs when the concentration of free ligand in the nucleus is 1 nM, the concentration of free T3 in the hepatic nucleus is also approximately 1 nM or about 200-fold greater than the concentration of free T3 in human serum, as measured in vitro by equilibrium dialysis. However, previous tracer kinetic studies have shown that T3 is available for transport from both the circulating albumin- and thyroid hormone-binding globulin-bound pools in plasma. Since the plasma proteins per se do not significantly exit the hepatic microcirculation on a single pass, the protein-bound hormone is operationally available for transport into liver via a proposed mechanism of enhanced dissociation caused by interactions between the plasma proteins and the hepatic microcirculation. These considerations raise the question as to whether the dissociation of T3 from serum proteins is sufficiently enhanced to allow for enrichment in the hepatic pools of exchangeable hormone that can generate 50% receptor occupancy. The present studies present a physiologically based steady state model of T3 transport and distribution in liver. All parameters of the model pertain to physiologically discrete pathways of hormone, plasma protein, or plasma flux through the liver. The rate constants were estimated from previously reported tracer kinetic transport data in rat liver in vivo using rat and human serum proteins. A computer program in BASIC for steady state analysis is presented. The results of the stimulation studies are as follows. The concentration of cellular exchangeable T3 in liver is predicted to be approximately 2 log orders greater than the concentration of free T3 measured in vitro, but approximately equal to the concentration of free T3 predicted to exist in the hepatic nucleus; thus, the high degree of hepatic nuclear T3 receptor occupancy is compatible with the model that T3 is available for transport into liver from the circulating serum protein-bound pools. The concentration of cellular exchangeable T3 is governed primarily by the concentration of plasma exchangeable T3 (and membrane transport and cellular metabolism of T3) and is predicted to be independent of changes in the concentration of cytosolic T3-binding proteins.(ABSTRACT TRUNCATED AT 400 WORDS)

Hyperthyroidism in children treated with long term medical therapy: twenty-five percent remission every two years.

We use an antithyroid drug for the treatment of hyperthyroidism due to Graves' disease in children and adolescents for as long as the patients are willing to comply and/or tolerate the drug. In more than 60 patients treated since 1961, the remission rate was 25% in the first 2 yr. This report looks at these same patients again, followed for an additional 5 yr. Survival analysis methods applied to the follow-up data on 63 children confirm our original statistical findings and suggest a continuing remission rate of 25% every 2.1 +/- 0.4 (+/- SE) yr regardless of the duration of previous therapy. The median time to remission was 4.3 +/- 1.5 yr, and 75% of patients are predicted to be in remission in 10.9 +/- 2.3 yr. Of 36 patients who went into remission, defined by their being euthyroid for 1 yr after cessation of therapy, 1 relapsed, and 2 developed spontaneous hypothyroidism; the remainder are euthyroid 1-11.7 yr after therapy was discontinued. Of 14 who switched from medical therapy, 2 of 7 treated surgically and 4 of 7 treated with 131I are hypothyroid. Only 1 patient had a significant adverse reaction to both methimazole and propylthiouracil. While medical therapy may have some direct effect on the autoimmune response in hyperthyroidism, its role in affecting the time to ultimate remission is unknown. These data, however, describe the course of children so treated and allow us to present therapeutic options initially or during treatment based on statistically derived probabilities of outcome.

Insulin binding to erythrocytes of normal infants, children, and adults: variation with age and sex.

The assay of insulin receptors on erythrocytes requires only small amounts of blood and has made it possible to characterize insulin binding in infancy and childhood. To establish normal insulin-binding criteria, we studied 125I binding to insulin receptors on erythrocytes from a large number of normal subjects, including 15 term deliveries, 45 prepubertal children (aged 2 months-12 yr), 15 adult women, and 15 adult men. Insulin binding to cord erythrocytes was significantly higher at tracer and physiological insulin concentrations than binding to cells from any other age group (P less than 0.001). In the prepubertal children after the newborn period, insulin binding was not related to age or sex and did not differ significantly from the binding to cells from adult women. Erythrocytes from adult males, however, bound significantly higher amounts of insulin than did those from adult women or prepubertal children at all insulin concentrations tested (P less than 0.01). Increased binding to cord erythrocytes appeared to be due to an increase in receptor affinity, while the increased binding in adult males was primarily a result of increased receptor concentration. The data confirm previous reports of increased insulin binding to fetal cells and indicate that erythrocyte insulin binding stabilizes at levels similar to those in adult females by the age of 2 months. The increased binding of insulin to erythrocytes from adult males compared to binding to erythrocytes from children or adult females suggests that androgens may increase erythrocyte insulin binding over prepubertal levels.

Carotid artery injection technique: bounds for bolus mixing by plasma and by brain.

Estimation of Michaelis-Menten kinetic parameters (Km, Vmax) of blood-brain barrier (BBB) transport processes with the carotid artery single injection technique assumes that mixing of the bolus with unlabeled substrate either from (a) circulating plasma or (b) amino acid efflux from brain, is minimal. The maximum extent to which the bolus could mix by these two sources is quantified in the present studies by measuring 14C-phenylalanine extraction in pentobarbital-anesthetized and conscious rats after the addition of 0-80% rat serum to the arterial injection solution. An upper bound (+/- SE) of bolus mixing due to mixing from both sources, expressed in terms of percentage of rat plasma, is 8.8 +/- 1.9 and 7.0 +/- 2.1% for the anesthetized and conscious rat, respectively. The estimated contribution to bolus mixing due to amino acid efflux from brain is 3.3 and 2.1% for the anesthetized and conscious rat, respectively. Based on these estimates, the upper bound for bolus mixing with circulating rat plasma is only 5.5 and 4.9%, respectively, for the anesthetized and conscious catheterized rat. Thus, any bolus mixing after rapid carotid injection is relatively small and is comparable to the mixing effects observed with the carotid artery infusion technique. Mixing effects on the order of 5% are shown to have no significant effect on the estimation of kinetic parameters of BBB nutrient transport, except for neutral and basic amino acid transport, which are characterized by very low Km values relative to the usual amino acid plasma concentrations.(ABSTRACT TRUNCATED AT 250 WORDS)

Interictal seizure resections show two configurations of endothelial Glut1 glucose transporter in the human blood-brain barrier.

Immunogold electron microscopy was used to analyze and quantify the Glut1 glucose transporter in brain tissue from five patients undergoing surgery for treatment of seizures. Samples were prepared from two different regions of each resection: (1) the most actively spiking epileptogenic site, and (2) the least actively spiking region, as indicated by intraoperative EEG monitoring. Two configurations of endothelial cell Glut1 were observed. About one half of the capillary profiles examined displayed abundant Glut1 immunoreactivity on both luminal and abluminal endothelial membranes. In the remainder of the profiles, reduced Glut1 labeling was seen, but adjacent erythrocyte membranes remained highly Glut1 immunoreactive, suggesting that reduced endothelial Glut1 reactivity was not attributable to method artifacts. Immunogold studies using antisera to human glial fibrillary acidic protein and human serum albumin demonstrated increased quantities of these two epitopes in the extravascular regions in which more EEG spiking activity had been demonstrated. These observations were consistent with the hypotheses that capillary integrity was more compromised, and gliosis was quantitatively increased, in the more actively spiking region of the resection. Altered glucose transporter activity in the blood-brain barrier was characterized by a bimodal Glut1 distribution in which the smaller (type B) endothelial cells displayed low Glut1 immunoreactivity, whereas adjacent (and even contiguous) larger (type A) endothelial cells showed 5- to 10-fold greater expression of membrane Glut1 transporter protein. Because this transporter facilitates glucose entry to the brain, small pericapillary volumes of brain tissue may have quite different concentrations of glucose. We hypothesize that in complex partial seizures and other forms of brain insult, an alteration of blood-brain barrier Glut1 glucose transporter activity is indicated by the appearance of these two subpopulations of endothelial cells. In comparison with previous studies of human brain capillaries in hemangioblastoma and brain injury, endothelial Glut1 density was apparently reduced (interictally) in affected temporal lobes of patients with complex partial seizures.

An analysis of a class of DNA sequence reading molecules.

Linked polyamides are a class of designed molecules that bind in the minor groove of double-stranded DNA in a partially sequence-specific manner but have limited sequence discriminatory abilities. This suggests a need for design alternatives to create molecules with enhanced sequence specificity. In this report we present formal proofs of the theoretical limits of the DNA sequence specificity of hypothetical sequence reading molecules as a function of their base recognition properties and sequence content and length of their target sequence. We prove that molecules containing nonspecific readers at critical positions within the molecule may have enhanced sequence specificity over molecules composed entirely of base specific reading elements. We also determine optimal patterns of base recognition for molecules in order to optimize their target sequence specificity. We also examine the effect of the length of a polyamide (i.e., the number of base pairs it binds) on its sequence discriminatory ability and determine necessary concentration dependent constraints on the binding free energies in order for longer polyamides to have greater sequence specificity than shorter ones. We show that unless the discriminatory ability of a ring for its preferred base is very strong, longer polyamides do not necessarily have greater sequence specificity over shorter ones when compared at the same molar concentration.

Thrombotic microangiopathy following allogeneic bone marrow transplantation is associated with intensive graft-versus-host disease prophylaxis.

Thrombotic microangiopathy (TM), manifesting clinically as thrombotic thrombocytopenic purpura or hemolytic uremic syndrome, is an uncommon complication after bone marrow transplantation (BMT). A retrospective analysis of potential risk factors for TM following allogeneic BMT was performed. Clinical data were analyzed from seven patients diagnosed with severe TM and 409 patients who underwent BMT during the same time period and who survived for at least 100 days afterwards. Six of the seven patients with TM received intensive GVHD prophylaxis consisting of cyclosporine, methotrexate and glucocorticoids, whereas only 66 of the 409 patients without TM received this regimen (P < 0.001, Fisher's exact test). This regimen was administered to patients older than 40 years, or recipients of a mismatched or unrelated allograft. Univariate analysis also revealed an increased risk of TM associated with the use of an unrelated bone marrow donor (P = 0.02), but no significant association with patient age or gender, diagnosis, amount of prior chemotherapy, transplant conditioning regimen or severity of GVHD. A multivariate exact logistic regression analysis revealed that only the type of GVHD prophylaxis had a significant impact on the risk for TM. The combined use of cyclosporine, methotrexate and glucocorticoids as GVHD prophylaxis may predispose to the development of TM following BMT.

13CO2 washout dynamics during intermittent exercise in children and adults.

To test the hypothesis that children store less CO2 than adults during exercise, we measured breath 13CO2 washout dynamics after oral bolus of [13C]bicarbonate in nine children [8 +/- 1 (SD) yr, 4 boys] and nine (28 +/- 6 yr, 5 males) adults. Gas exchange [O2 uptake and CO2 production (Vco2)] was measured breath by breath during rest and during light (80% of the anaerobic threshold) intermittent exercise. Breath samples were obtained for subsequent analysis of 13CO2 by isotope ratio mass spectrometry. The tracer estimate of Vco2 was highly correlated to Vco2 measured by gas exchange (r = 0.97, P < 0.0001). The mean residence time was shorter in children (50 +/- 5 min) compared with adults (69 +/- 7 min, P < 0.0001) at rest and during exercise (children, 35 +/- 7 min; adults, 50 +/- 11 min, P < 0.001). The estimate of stored CO2 (using mean Vco2 measured by gas exchange and mean residence time derived from tracer washout) was not statistically different at rest between children (254 +/- 36 ml/kg) and adults (232 +/- 37 ml/kg). During exercise, CO2 stores in the adults (304 +/- 46 ml/kg) were significantly increased over rest (P < 0.001), but there was no increase in children (mean exercise value, 254 +/- 38 ml/kg). These data support the hypothesis that CO2 distribution in response to exercise changes during the growth period.

First-pass effect of an intravenous bolus of [13C]bicarbonate displayed breath-by-breath.

The dilution of an intravenous bolus dose of [13C]bicarbonate is used as an estimate for the metabolic rate under certain conditions. It is a consistent finding in all studies that the total amount of intravenous [13C]bicarbonate cannot be recovered as breath 13CO2. In this study, we used a breath-by-breath analysis of 13CO2 to depict the washout of 13CO2 at a high temporal resolution to analyze the extent to which a probable first-pass effect is responsible for the reduced recovery. Eight healthy men were tested at seated rest and with bicycle exercise at a constant load relative to 40 and 75% maximal O2 consumption VO2 max). [13C]bicarbonate (0.0125 g/kg body wt) was administered as an intravenous bolus in each test. Respiratory mass spectrometry was used to derive the course of the end-tidal 13CO2-to-12CO2 ratio from the breath-by-breath data. Approximately 2 min after 13C administration, the washout curve could be fitted well by a two-exponential curve describing a two-compartment mammillary model. Immediately after administration of the bolus dose, an excess peak in the end-tidal 13CO2-to-12CO2 ratio appeared. This peak could not be included in the two-exponential fitting. The area under the first peak resulted in 3.8 +/- 1.3% of the total [13C]bicarbonate dose at rest, 11.5 +/- 2.9% at moderate exercise (40% VO2 max), and 16.9 +/- 4.0% at intensive exercise (75% VO2 max). The first-pass effect had an increasing impact of up to about two-thirds of the lacking bicarbonate with higher exercise intensity. The "loss" of tracer via this first-pass effect must be considered when the results of studies with parenteral administration of [13C]bicarbonate are considered, especially when it is given as a bolus dose and during exercise.

Oral [13C]bicarbonate measurement of CO2 stores and dynamics in children and adults.

During exercise, less additional CO2 is stored per kilogram body weight in children than in adults, suggesting that children have a smaller capacity to store metabolically produced CO2. To examine this, tracer doses of [13C]bicarbonate were administered orally to 10 children (8-12 yr) and 12 adults (25-40 yr) at rest. Washout of 13CO2 in breath was analyzed to estimate recovery of tracer, mean residence time (MRT), and size of CO2 stores. CO2 production (VCO2) was also measured breath by breath using gas exchange techniques. Recovery did not differ significantly between children [73 +/- 13% (SD)] and adults (71 +/- 9%). MRT was shorter in children (42 +/- 7 min) compared with adults (66 +/- 15 min, P less than 0.001). VCO2 per kilogram was higher in the children (5.4 +/- 0.9 ml.min-1.kg-1) compared with adults (3.1 +/- 0.5, P less than 0.0001). Tracer estimate of CO2 production was correlated to VCO2 (r = 0.86, P less than 0.0001) and when corrected for mean recovery accurately predicted the VCO2 to within 3 +/- 14%. There was no difference in the estimate of resting CO2 stores between children (222 +/- 52 ml CO2/kg) and adults (203 +/- 42 ml CO2/kg). We conclude that orally administered [13C]bicarbonate can be used to assess CO2 transport dynamics. The data do not support the hypothesis of lower CO2 stores under resting conditions in children.

Unrelated donor bone marrow transplants in children.

Only a small proportion of children who might benefit from bone marrow transplant (BMT) have an HLA-identical sibling. To provide this potentially curative therapy to patients without a matched related donor, marrow transplants using less well matched related donors or unrelated donors (identified through computerized donor registries) have been performed. We report the outcome of 24 consecutive unrelated donor BMT's performed on children. Eligible diagnosis included acute leukemia (AL) (n = 15), chronic myelogenous leukemia (CML) (n = 4), myelodysplastic syndrome (MDS) (n = 3), and severe aplastic anemia (SAA) (n = 2). All donor/recipient pairs were sero-matched at 5 or 6 of the 6 HLA A, B, and DR antigens. Several different preparative regimens were used, but fractionated total body irradiation (TBI) was used in 20 patients. All recipients received graft-versus-host-disease (GVHD) prophylaxis with cyclosporine-A (CSA), four with short course methotrexate (MTX), 14 in combination with short course MTX and methylprednisolone (MPS), and five in combination with a mouse monoclonal antibody to CD5, coupled to the A-chain of ricin (Xomazyme-65). One patient received CSA and MPS alone after a T-cell depleted marrow transplant. Twenty of 23 evaluable recipients engrafted (87%). Two patients with CML never engrafted and had autologous marrow recovery, one patient with SAA died at 128 days without evidence of engraftment, and there was one early death at day + 9. Fourteen of 20 patients (70%) with stable donor-derived hematopoiesis developed significant acute GVHD > or = grade II). Eleven of 15 engrafted patients who survived > 100 days after BMT developed chronic GVHD (73%).(ABSTRACT TRUNCATED AT 250 WORDS)

Comparison of tolmetin kinetics in rheumatoid arthritis and matched healthy controls.

We could find no significant differences in the kinetics of tolmetin when comparing five rheumatoid arthritis patients with moderate disease activity with a carefully matched group of normal healthy volunteers. Further, there were no discernible clinically significant differences in the kinetics of tolmetin when comparing a single dose to one week of therapy. These results, although limited by the small number of subjects involved and the large interpatient variability, suggest that it may be possible to extrapolate pharmacokinetic data from normals to patients with moderately active disease.

Intracisternal injection of TRH precursor, TRH-glycine, stimulates gastric acid secretion in rats.

Intracisternal injection of thyrotropin-releasing hormone (TRH)-Gly (pGlu-His-Pro-Gly) produced a dose-dependent (1-100 micrograms) stimulation of gastric acid secretion in urethane-anesthetized rats implanted acutely with a gastric fistula. The peak response occurred 20-30 min after intracisternal injection and lasted for more than 2 h. Intravenous injection of TRH-Gly (100 micrograms) did not modify gastric acid secretion. Following intracisternal injection of TRH-Gly, a peak elevation of both TRH-Gly and TRH levels is observed in the cerebrospinal fluid (CSF) within 15 min. Thereafter, TRH values are returned to basal levels at 75 min after the injection, whereas TRH-Gly concentrations remain significantly elevated throughout the 2-h period of measurement. Compartmental analysis revealed that CSF conversion of TRH-Gly to TRH was only 0.0072%/min. Medullary coronal sections containing the dorsal vagal complex and the raphé nucleus revealed increased content of TRH-Gly, but not TRH, 40 min after administration of TRH-Gly at an intracisternal dose effective in stimulating gastric acid secretion (100 micrograms). In addition, TRH but not TRH-Gly (10(-7)-10(-5) M) displaced [3H]MeTRH binding from rat medullary blocks containing the dorsal vagal complex. These data suggest that the intracisternal TRH-Gly-induced stimulation of gastric acid secretion is not related to its conversion to TRH in the CSF, or direct activation of TRH receptors in the medulla. The acid secretory response of TRH-Gly may be due to the formation of TRH at the active brain sites, or alternatively to activation of its own specific receptors.

Developmental modulation of blood-brain-barrier glucose transport in the rabbit.

Blood-brain barrier (BBB) glucose transport rates were measured using the intracarotid injection method in newborn, 14-day-old suckling, 28-day-old weanling and adult rabbits, and compared with membrane transporter density. Light microscope immunochemistry confirmed the presence of the GLUT1 glucose transporter isoform in these rabbits. Quantitative electron microscopic immunogold analyses of GLUT1-immunoreactive sites per micrometer of capillary membrane indicated GLUT1 density increased with age, and correlated with in vivo measurements of Vmax. Maximal transport velocities (Vmax) of glucose transfer (an indicator of the activity and relative number of transporter proteins) increased significantly (P = 0.05) with age: in neonates Vmax = 0.61 mumol.min-1.g-1, in sucklings Vmax = 0.68 mumol.min-1.g-1, in weanlings Vmax = 0.88 mumol.min-1.g-1, and in adults Vmax = 1.01 mumol.min-1 g-1. Cerebral blood flow (CBF) rates, increased with age from 0.19 and 0.26 ml.min-1.g-1 in neonates and sucklings to 0.51 (weanlings) and 0.70 (adults) ml.min-1.g-1. Non-linear regression analyses indicated the half-saturation constant (Km) for glucose transport ranged from 13 mM in adult rabbits to 19 mM in 14-day-old sucklings: differences in Km were not significant. Age-related changes in the Permeability-Surface Area product (PS +/- S.E.) of both water and glucose were also seen. At all ages studied, the diffusion component (Kd) of glucose uptake was not distinguishable from zero. We conclude developmental up-regulation of the rabbit BBB glucose transporter is characterized by no changes in transporter affinity, and provide the first demonstration of increased membrane transporter proteins correlating with an age-related increase (65%) in glucose transporter maximal velocity.

Persistence of insulin resistance in polycystic ovarian disease after inhibition of ovarian steroid secretion.

Six nonobese women with polycystic ovarian disease (PCOD) showed significant hyperinsulinemia, compared with controls after oral glucose (P less than 0.05). As an indicator of insulin sensitivity, in vitro proliferation of erythrocyte progenitor cells of PCOD subjects exposed to physiologic concentrations of insulin was significantly blunted (P less than 0.001). Monocyte insulin receptor binding was not impaired in the PCOD subjects. Three of the PCOD patients were treated with a long-acting gonadotropin-releasing hormone agonist for 6 months, which resulted in marked suppression of ovarian androgen secretion but no demonstrable changes in in vivo or in vitro indicators of insulin resistance. Thus insulin resistance in PCOD subjects appears to be unrelated to ovarian hyperandrogenism (or acanthosis or obesity). Although certain tissues are insulin-resistant in PCOD patients, the ovary may remain sensitive and overproduce androgens in response to high circulating insulin levels.